Nerve growth factor has a modulatory role on human primary fibroblast cultures derived from vernal keratoconjunctivitis-affected conjunctiva.

PURPOSE
To evaluate the role of nerve growth factor (NGF) in remodeling processes of vernal keratoconjunctivitis (VKC). VKC is a chronic inflammatory disorder of the conjunctiva and is characterized by marked tissue remodeling. NGF, a pleiotrophic factor with documented profibrogenic activities, is produced by inflammatory and structural cells populating the VKC conjunctiva and is increased in the serum and tears of VKC patients.


METHODS
Primary cultures of VKC-derived fibroblasts (VKC-FBs) were exposed to increasing NGF concentrations (1-500 ng/ml) to evaluate and compare the expression of alpha-smooth muscle actin (alphaSMA, a defining myofibroblast marker), collagens (types I and IV), and metalloproteinases and tissue inhibitors (MMP9/TIMP1, MMP2/TIMP2) at the biochemical as well as molecular levels.


RESULTS
Endogenous NGF was increased in the VKC-FB supernatant, as compared to healthy-FB supernatant. VKC-FBs expressed alphaSMA and increased types I and IV collagens. VKC-FBs, and in particular all alphaSMA positive cells, expressed both trkA(NGFR) and p75(NTR), while healthy-FBs only expressed trkA(NGFR). Exogenous NGF did not change alphaSMA expression, while alphaSMA expression was enhanced by specific neutralization of p75(NTR). NGF (10 ng/ml) exposure significantly decreased type I collagen expression, without affecting type IV collagen, and increased MMP9mRNA and protein.


CONCLUSIONS
The autocrine modulation of differentiation and response of VKC-FBs to NGF exposure with downregulation of type I collagen and upregulation of MMP9 expression supports a relevant role for NGF in tissue remodeling of VKC.

age 12.67±2.08) and sex/age matched healthy (n=3) conjunctiva. Informed consent was given by the parents of each patient and the approval of the Intramural Ethic Committee was granted, in conformity to the Declaration of Helsinki. The diagnosis of active VKC was based on history, clinical examination and the presence of eosinophils in conjunctival biopsy. Clinical scores (0-3: 0, absent; 1, weak; 2, mild; 3, severe) for each ocular symptom (itching, tearing, photophobia and foreign body sensation) and each sign (conjunctival hyperemia, mucous discharge, papillae and corneal epithelial erosion) were assigned at the time of examination. Total symptom (range 0-3) and sign scores (0-12) were calculated showing: total symptom score of 7.33±4.51 and total sign score of 8.67±3.05. The three patients showing lid-ptosis were submitted to surgical removal of the papillae that were processed in this study.
Relative real-time RT-PCR: Total RNA (1x10 6 cells) was extracted in OMNIzol (Euroclone), DNaseI treated (AB1709; Ambion Inc., Austin, TX), quantified (λ 260 /λ 280 >1.8) and checked for RNA integrity. Three µg total RNA were reverse ). Product specificity was assessed by melting curve analysis of each sample carried out in duplicate as well as by gel size-fractioning. C t values normalized samples showing good melting curves were used for statistical analysis. Differences in PCR product expression were evaluated by REST© software [13].
Proliferation assays: The effect of increasing NGF concentration on VKC-FB proliferation was investigated by counting the cells, after brief enzymatic digestion, using the trypan blue exclusion test. The expression of the nuclear proliferating factor ki67, recognizing all the cell cycles except G0 [14], was investigated on previously fixed monolayers stained with rabbit antihuman ki67 antibody (1:1000, Santa Cruz) and developed by fluorescent ABC technique (Vector).
SDS-PAGE zymography: After culturing, the conditioned media were collected and clarified by centrifugation. MMP activity was analyzed by zymography, using a procedure described in reference [18]. Briefly, 50 µl conditioned media were mixed with SDS sample buffer, without β-ME, and heated for 37 °C/30 min. Normalized samples (30 µg/lane; Bio-Rad protein assay) underwent standardized electrophoresis in 10% SDS-PAGE containing 0.1% gelatin (Bio-Rad). Molecular weight markers (6-210 kDa), recombinant human latent MMP9 (92 kDa), and active MMP9 (83 kDa; Calbiochem) were loaded as positive controls. The gel was washed in 2.5% TX-PBS to remove SDS and renature the proteins, incubated at 37 °C for 48 h in an activation buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl 2 ; pH 7.5), rinsed in ddw, and finally stained for 60 min with 0.25% Coomassie brilliant blue R250 in 40% isopropanol. Gelatinolytic activity was identified as clear bands on a uniform blue background following destaining in 7% acetic acid, indicating the area where gelatin was digested.
Statistics: All experiments were done three times, with each point carried out in duplicate. Experimental results are expressed as mean±SEM. Parametric ANOVA followed by Tukey-Kramer post hoc was employed to analyze the data [19], using the statistical package StatView II for PC (Abacus Concepts Inc., Barkley, CA). A probability of less than or equal to 0.05 was considered to be statistically significant.

RESULTS
Vernal keratoconjunctivitis-derived fibroblasts express nerve growth factor, trkA NGFR , p75 NTR , and αSMA: Increased NGF levels were detected by specific ELISA in the conditioned media of VKC-FBs and compared to healthy-FBs (563.33±35.12 pg/ml versus 445.67±10.59 pg/ml; 26.51% increase; p=0.005). This data is in accordance with the NGF mRNA upregulation observed in VKC-FBs ( Figure 1A). RT-PCR and confocal analysis revealed that VKC-FBs expressed both trkA NGFR and p75 NTR . In contrast, healthy FBs only express trkA NGFR , as previously reported [8] (Figure 1A,B). A slight downregulation of trkAmRNA expression was found in VKC-FBs, in comparison to healthy ones (p<0.05). As detected by cs-ELISA, a significant increase of αSMA expression was observed in VKC-FBs (0.378±0.027 OD VKC-FBs versus 0.232±0.046 OD healthy-FBs; a 1.62-protein increase [p<0.01]). VKC-FBs expressed αSMA protein in association with p75 NTR (Figure 1C). No proliferation was observed in the presence of increasing NGF concentrations (data not shown).
Nerve growth factor does not modulate αSMA expression: VKC-FBs were exposed to increasing NGF concentrations (1-500 ng/ml) in order to investigate whether NGF was able to influence αSMA expression. At both cs-ELISA and western blot analysis, NGF was not able to influence αSMA expression in VKC-FBs (Figure 2; dark bars [p>0.05]). NGF stimulation did not influence αSMA expression after preincubations with neutralizing trkA NGFR antibodies, (data not shown). In contrast, preincubation with neutralizing p75 NTR antibodies followed by increasing NGF concentrations resulted in an in-983 crease of αSMA expression (Figure 2; light bars [p<0.05]). The pattern of αSMA expression resembled those of NGFtreated healthy FBs [8].
Nerve growth factor modulates types I and IV collagen expression: In VKC-FBs, the high amount of αSMA was found associated with a high rate of types I and IV collagen (rate; 7.78: type I, 1.323±0.248 and type IV, 0.170±0.004), as compared to healthy FBs (type I, 0.993±0.001 and type IV, 0.077±0.014; p<0.05; see also Table 1). After NGF exposure, the expression of type I collagen by VKC-FBs was significantly decreased at all the concentration levels tested ( Figure  3A). The maximum effect was observed at 10 ng/ml (1.33target gene decrease, p<0.05), reaching a level almost comparable to those of healthy-FBs ( Figure 3A; light bar). By contrast, the expression of type IV collagen was not changed at any NGF concentration level ( Figure 3B; light bars).
Nerve growth factor modulates MMP-9 expression and function: The expression and functional activities of MMP9, known to drive specifically collagen type IV cleavage [20], was investigated as a function of NGF stimulation in VKC-FBs. VKC-FBs were exposed to increasing NGF concentra-  (Cy2, green). p75 NTR and trkA NGFR colocalized in some cellular compartments (merge; X600/oil immersion). C: Confocal microscopy on VKC-FBs showing, from left to right, that these cells express p75 NTR (Cy3, red) and αSMA (Cy2, green). p75 NTR and αSMA colocalized in some cellular compartments (merge; X600/oil immersion). tions (1-100 ng/ml) and the expression of MMP9 protein and mRNA were evaluated and compared to untreated VKC-FBs. By western blot analysis, it was determined that NGF significantly increased MMP9 protein expression in VKC-FBs, as compared to healthy-FBs, in a dose-dependent fashion (Figure 4A). This specific MMP9 increase was associated with an increase of MMP9 activities, as shown by gelatinolytic bands, at the same concentrations tested ( Figure 4B). In agreement with biochemical data, the molecular analysis showed that NGF was able to trigger the MMP9-mRNA expression in a dose-dependent fashion with the maximum expression at 10 ng/ml NGF (3.92-target gene increase, p<0.05; Figure 4C). Interestingly, the MMP9-mRNA increasing effect of 10 ng/ ml NGF was found to be comparable to that of 1 ng/ml TGFβ1 (5.48±0.04 versus 7.49±0.09, respectively). According to the literature, MMP9 mRNA was found to be increased in VKC-FBs as compared to healthy-FBs (24.98-target gene increase; p<0.05 [3]). Biochemical and molecular characteristics of healthy and VKC-derived FBs are summarized in Table 1.

DISCUSSION
Our data demonstrated that primary cultures of VKC-FBs express both trkA NGFR and p75 NTR receptors and produce high levels of NGF, types I and IV collagens, and MMP9. Moreover, VKC-FBs are mainly represented by myoFBs, since a consistent proportion of VKC-FBs were αSMA positive.
Various Th2-derived cytokines and growth factors are increased in blood, tears, and conjunctiva from VKC patients. These profibrotic factors were found able to drive the crosstalk between structural (epithelium and FBs) and inflammatory (lymphocytes, eosinophils, and mast cells) cells. This cellfactor interaction contributes to the chronic inflammatory process, giant papillae formation and tissue remodeling, as observed in VKC [2,3,5,[21][22][23]. Among these, TGFβ1 isoform remains the main pro-fibrogenic factor, being responsible for ECM ex-novo deposition, the inhibition of ECM degradation and the prolonged myoFB activity [24].
NGF is also increased in VKC blood and conjunctiva as a result of the activation of both structural (epithelium and FBs) and inflammatory cells (Th2 lymphocytes, mast cells, and eosinophils), during the active conjunctivitis [7,22]. Older and more recent data [8,[22][23][24] indicate that NGF is a pleiotrophic factor participating to the control of inflammatory responses, tissue repair, fibrosis, and remodeling in different tissues. Primary cultures of healthy-FBs have been found to be modulated by NGF with relation to cell migration, differentiation, and contraction of a cell matrix [8].
Since stromal FBs represent the major target/effector cells involved in tissue remodeling [6,12,25] and since NGF activates in vitro healthy-FBs [8,26,27], we sough to evaluate the possible modulation of FBs isolated from conjunctiva of patients with VKC by NGF, hence no data are available in literature.

TABLE 1. BIOCHEMICAL AND MOLECULAR DESCRIPTION OF HEALTHY AND VERNAL KERATOCONJUNCTIVITIS-DERIVED FIBROBLASTS
Biochemical and molecular description of healthy and vernal keratoconjunctivitis-derived fibroblasts In the present study, increased expression of NGF, associated with increased expression of p75 NTR and αSMA, was detected in VKC-FBs as compared with conjunctival healthy-FBs. In addition, αSMA was found expressed in FBs showing light upregulation of trkA NGFR and considerable upregulation of p75 NTR , suggesting a specific role for NGF in VKC-FBs. In line with the effect of NGF in driving the differentiation of healthy FBs into myoFBs [8], we wondered whether NGF supplementation to VKC-FBs cultures would result in a further VKC-FB differentiation, evaluated as αSMA expression. Interestingly, NGF failed to further increase αSMA expression in VKC-FBs, unless p75 NTR was blocked (see Figure 2; light bars). This data is of great interest, since it suggests two possible hypothesis: (1) NGF plays a differentiating effect through the specific and unique binding to trkA NGFR (NGF is a specific receptor), but not p75 NTR (the pan-neurotrophin receptor); and (2) the specific expression of p75 NTR , by myoFB phenotype (otherwise absent in FBs), seems to play a switchoff effect in the further differentiating action of NGF, suggesting that NGF plays a modulatory rather than a exclusive stimulatory effect on the fibrotic process. The answer might lie in trkA NGFR /p75 NTR multifaceted functions [7,22].
On the other side in VKC tissue remodeling process, NGF might modulate collagen production and might influence MMP2/MMP9 production/activity, as previously demonstrated for other growth factors [2,4]. ECM metabolism is heavily impaired in VKC, due to a substantial increase in total collagen deposition in the conjunctiva (mainly types I, III, and IV), and an increased release of MMP2/MMP9 in the tears [3,4]. In this study, the expression of type-I collagen decreased significantly after NGF exposure, rather quite specific given that this effect was not observed for type-IV collagen or with the addition of TGFβ1 (data not shown). In addition, NGF was able to induce specifically MMP9 expression/activity by VKC-FBs at both molecular, biochemical and functional levels. This effect was not observed in healthy-FBs, as previously reported [8].
Taken together, these data suggest a many-sided role of NGF in VKC tissue remodeling. NGF is increased in VKC blood and tarsal conjunctiva and likewise in tears [9]. In in vitro studies, NGF induces the differentiation of healthy conjunctival FBs into myoFBs, the main effector and target cells of fibrotic process. This differentiating effect was not observed in VKC-derived conjunctival FBs in the present study. Additionally, NGF induced a decrease in type I collagen and an increase in MMP9 expression by VKC-FBs with no specific effect on both MMP1 and MMP2 mRNA expression. It has been previously reported that both MMP2 and MMP9 degrade types IV and V collagen; MMP9 can also degrade types I and type III collagen [28,29]. One possible explanation for these data might be related to the well-known singular action of NGF due to changes in the balance between trkA NGFR /p75 NTR expression. In this study we demonstrated a role of trkA NGFR in the differentiating effect of NGF on FBs; however, the role of p75 NTR on VKC-FB function seems complex and remains to be elucidated.
In VKC, various growth factors and Th2-cytokines are produced by inflammatory/stromal cells. In line with other studies, these factors have been proposed to modulate tissue remodeling in VKC. To summarize, our in vitro findings showing the NGF modulation of both type I collagen and MMP9, might propose NGF as an active contributor in VKC remodeling. Figure 4. Nerve growth factor increases MMP9 expression and function by vernal keratoconjunctivitis-derived fibroblasts. Conditioned media were collected and processed as described in Methods. A: The histogram shows a significant increase of MMP9 protein expression in VKC-FBs treated with increasing NGF concentrations, according to the densitometric analysis (mean OD±SEM; p<0.05). Data were normalized to GAPDH expression and presented as fold increase with respect to untreated VKC-FBs. B: The functional activity in MMP9 was investigated by SDS-PAGE zymography. From left to right (1-6 lines): 0, 1, 10, 100, 250, 500 ng/ml NGF; line 7, αNGF+100 ng/ml NGF. Panel represents one of three independent gels that gave the same results. C: Relative real-time PCR showed a significant increase of MMP9 mRNA expression in VKC-FBs treated with different concentrations of NGF (p<0.05). Data were normalized to GAPDHmRNA expression and presented as fold increase [11] with respect to untreated VKC-FBs. 986